Microbiological production of esters



,. AB '*QFZ HED SCLQSU E Fermentation process for the production ofesters w ch comprises fermenting an aliphatic hydrocarbon with Micraco ccus cerificans in a suitable growth medium characterized by c ontainingless than the amount required for maitimum growth of. mineral, nutrientsselected fretn th group consisting of divalent magnesium cation and di:valentcalcium cation 1.

This invention relates to a process for. biosynthetically producingesters from.,hydro carbons. Inparticular, this invention relates toconverting hydrocarbons to fatty acid esters by. the metabolic action ofmicroorganisms; More objects of 'tliisinven tioii .will'be more clearlyperceived and" understood by particularly, this invention relates to thebiosynthesis ,of

waxy-esters from hydrocarbons by aerobicallyflsubjecting ahydrocarbon tothe metabolic actionofa, gram negative bacteria in the presence ofan'aqueous mineral-saltssolution containing a limited.coneentrationof,mineralhwtrients. selected from .the group consistingof magnesium, calcium,,and .a combination thereof. Stillmoreparticularly, this invention relates to.the-utilization ofazelaic acidto Stimulate .said microbiological production ;.of esters. i It is knownthat microorganisms can be -aerob i cal1y cultivated on inexpensivehydrocarbon feed s tocks in-the presence of a non-limiting, aqueousinorganic salt growth medium in a suitable fermentation reactor. It isfurther known that as the hydrocarbon is assimilated by thegallic-wmilounasi er aldehyde'sr a19bhlsrester$- preferred process. forpurifying the hydrocarbon feed etc. arefor'medas by-products;Various-methods have" been proposed which favor microorganism cellroduction r and restrict by-product formation and vice versa. However,there is a constant-need for new and economic methods which result inthe selective formation of one prod not or another.

It is, therefore, an object of the present invention to provide ar'ticess fo r the biosyntlietic productioh of esters from hydrocarbons.

Another object of the present invention is to provide a process forstimulating the biosynth'etic production of esters fromhydroearbonsg" ki 'According to the present invention, it has'lnow'been discovered thatif the "aqueous'mineral saltsfsolution in which a, microorganism isaerobically cultivatedeontains a limited concentration, i.e., below thatamount needed; forunaitimum growth of the microorganism,ofthe min eralnutrients selected from the group consisting of'tn nesiumfcalciurn-and acombination thereof, byj-product formation of esters, is favored attheexpense of m i;cr'o-f organism cell growth. More specifically,' itjlias' been found that if the concentration of'Qthe' divaleiit magnesium'(Mg++) cation in the aqueous' 'mine'ral salts solution is restricted tonot more than about'002 wt."percent,"based on the total amount of theaqueou's 'g'rowth medium and/ 01 if the eo'ncent'rationofthedivalentcalcium (Ca cat'ion'is restricted-to not more-than about 0.0-1-w't;-"per-' cent, based on total amount ofthe aqueous growth-me:vdium,-the' amount of ester by-product is significantlyincreased iat'theexpense lof cellular growth. It has furtherbeenafound that'the use of.small...amounts, e.g., between referring to the following descriptionand claims. HydrOcarbOnfeeds which can' be utilized'ior" the presenf'processf are Cf-Cg petroleum; hydrocarbon feeds, preferably'gas-oilsboiling in the range of between about 190 C. and about 400 Cpreferably'"between abbiit 190- C? and about320" C. "Other suitablefeeds are C1-C normal and isoparaffins, cycloparafiins,- monoolefins',"diolefin's, aromatics and-mixtures thereof. Feeds available inlargdqu'ant'ities 'and those particularly suitable meo -c normalpa-raifi'ns from- -gas oils, light naphthas, and normally gaseousfeedsfsuch as methane, ethane; propane; butane' an'dmixtures-thereof,'e.g., hatural gas. Where'normally gaseous feeds'areused, these are, of course; preferably supplied directly to the reactorthrough spargers submergedin the liquid mediumA further pre ferred feedis one which contains a substantial weight percentage, e.g., 70+ weightpercent,'of normal -(straight chain) parafiin hydrocarbons having from1-0 to 30-carbon atoms. While the presence of branched, non-aromatichydrocarbons in amounts of up to 30% by weight in the hydrocarbon feedcan-be tolerated, c'encentrations'in eitcess-of 10 weight percent ofnon-normal, non-aromatic hydrocarbons are usually avoided since thepreferred microorganisms employed in the present process are selectiveto normal hydrocarbons, especially intermediate ra'nge (C C n-paratfins.Therefore, the use of branched parafiins is preferably avoided.

A most preferred hydrocarbon feed is a C -C feed stock which has beenpurified .to reduce the level of aro- 'rnatics, both plynuclear andmononucleanto below about 0.5 weight percent, preferably below about 0.1Weight percent, more preferably below about 100 ppm. This is necessarysince it has been discovered that organisms and aclean-up of-thedesorbed normal hydrocarbons with 13X sieves or silica gel to adsorbremaining impurities, particularly aromatics. One such. purificationprocess is described in US. Patent 3,070,542, which is incorporatedherein by reference. The essence of the disclosure of this patent is thefinding that by preloading the molecular sieve by the displacing medium,preferably ammonia, and by introducing a displacing medium along withthe feed, the rate of adsorption is increased and subsequent desorptiongreatly eased, particularly with high molecular weight materials. Theclean-up step of the desorbed normal hy-. droca rbons can be performedin a manner described in co-assigned US. patent applications Ser. No.223,078, filed Sept. 12, 1962, now US. Patent 3,228,995 issued Jan. 11,1966, or Ser. No. 223,057, filed Sept. 12, 1962, now US. Patent3,233,003. issued Feb. 1, 1966, which specifications are alsoincorporated-.herein by reference.

The purified hydrocarbon feed usually contains-about weight percent of C-C n-paralfins and .up to about 10 weight percent of n-olefins. Thepreferred purified hydrocarbon feed contains about weight percent of .C-C n-paraffins and up to about 5 weight percent i normal olefinscontaining from 1.1 to 30 carbon atoms.

mentation reactor (including the recycle), based on total aqueous growthmedium supplied, is between about 0.1 and about 10 weight percent,preferably between about 0.5 and about weight percent and especiallybetween about 0.5 and about 2 weight percent when straight air is usedas the oxygen carrying medium. When using oxygen-enriched gases, e.g.,gases having 70+ weight percent oxygen, a preferred amount ofhydrocarbon supplied to the reactor is between about 0.5 and about 5.0weight percent, based on the total equeous growth medium. The weightpercent of, for example, C -C normal hydrocarbons actually existing inthe slurry zone of the continuous reactor during by-product formationcan range from between about 0.01 and about 1.0 weight percent,

Especially preferred is Micrococcus c'erificans, isolated and identifiedby Dr. R. E. Kallio et al., Journal of Bacteriology vol. 78, No. 3,pages 441-448 (September 1959). Cultures of this organism have beendeposited in the American Type Culture Collection, 212 M Street,Northwest, Washington 7, D.C. The full identification of this materialis as follows:

Morphology: Cells are small, spherical, tending to be elliptical in oldcultures and in media high in nitrogen. Cells from defined media average0.5 to 1.0 in diameter, from complex media cell diameters 1.0 to 2.0a.Cells occur singly or in clumps. Immotile, metachromatic granules andsudanophilic granules are not observed.

Gramreaction: Negative.

but usually ranges between about 0.01 and about 0.1, 15 Colonies ondefined agar are small (1 mm), circular, preferably between about 0.01and about 0.05 weight convex, having entire edge. Colonies on nutrientagar are percent. larger (2 to 5 mm.) raised mucoid, generally round.Microorganisms which can be used in the present pro- Pigmentation:White, beige or tan variants occur. cess include any aerobic, bacteriacapable of assimilating Obligately aerobic. A wide variety of materialssup- C C hydrocarbons. Especially preferred are gram negports growth,yeast extract, casein hydrolyzate, long-chain ative bacteria whichassimilate paraffinic hydrocarbons. alchohols and acids, long-chainnormal alkanes and ole- While the present invention is applicable to abroad scope fins. of operable microorganisms, there are nine microorgan-Carbohydrate fermentation: No carbohydrates are ferisms which areespecially suitable for hydrocarbon asmented. Aerobically, manycarbohydrates are assimilated. similation. These microorganisms aretabulated herein- These include glucose, maltose, mannitol, sucrose,lactose, below along with their corresponding A.T.C.C.regisarabinose,rhamnose, sorbitol, dulcitol, and inulin. Aerobtrationnumbers, which were secured by depositing ically, glucose is utilizedwith acid production. Gluconic samples with the American Type CultureCollection in acid has been identified. Washington, D. C. Nitratereduction: Negative. Gelatin liquefaction: Generally negative. Slowliquefaction may occur in some Microorganism name? numb strains. Ureahydrolysis: Negative or slow hydrolysis. Micrococcus cerificans 14987Catalase is produced. Hydrogen is not utilized. Optimal Pseudomonas Iiustri 15522 temperature is 25 C. Optimal growth pH is 7.0 to 8.5.Pseudomonas pseudomallei 15523 Source: Iowa soil. Habitat: Soils.Pseudormonas orvilla 15524 It is noted that a more recent identificationshows the Alealigenes sp. 15525 organism is probably an Arthrobacterrather than a Mi- Cellumonas galba 15526 crococcus and closely resemblesArthrobacter ureafaciens. Brevibacterium insectiphilium 15528 Thefollowing summary of products indicates the reasons Corynebacterium sp.15529 for the preferred identification of this organism as anColynebacterium pourometabolum 15530 Arthrobacter:

Micrococcus M. Cerificans Arthrobacter Always gram positive Always gramnegative. Gram negative or variable.

early in fermentation.

quently fermented.

It is also to be understood that the particular class and subclass ofbacteria utilized is determined by the particular feed employed. Forexample, when the microorganisms assimilate methane or other gaseousparaffin feeds, the preferred class of microorganisms isPseudomonadaceae, such as Pseudomonas methanica. When fermentation isperformed using a light naphtha feed, the preferred classes ofmicroorganisms are Pseudomonadaceae and Arthrobacter, such asPseudbmonas fluorescens, Pseudomonas desmolyticum, Pseudomonasaeruginosa and Arthrobacter globiforme.

In a preferred embodiment, the biosynthesis is conducted using abacteria inoculant, especially gram negative coccus bacteria.

While any aerobic bacteria cells capable of assimilating normal C to Chydrocarbon feeds can be employed, preferred bacteria are as follows:Micrococcus cerificans (Arthrobacter ureafaciens), Pseudomonasaeruginosa, Pseudombnas fluorescens, Nocardia opaca, Nocardia rubra,Nocardia coralina, Pseudomonas methanica, Pseudomonas desmolyticum andMycobacterium phleie,

Oxygen is Supplied to the fermentation reactor in any form capable ofbeing assimilated readily by the inoculant microorganism.Oxygen-containing compounds can be used as long as they do not adverselyaffect ester production. Conveniently, oxygen is supplied as anoxygen-com taining gas, e.g., air, which contains between about 19 andabout 22 mole percent oxygen. While it is preferable to employ air,oxygenenriched air having more than 22 mole percent oxygen can be used.In general, between about 0.1 and about 10, preferably between about 0.5and about 4.0, more preferably between about 0.8 and about 2.5, volumesper minutes of air are supplied to the reactor per volume of reactorliquid.

Nitrogen is also supplied to the fermentation reactor. The source ofnitrogen can be any organic or inorganic nitrogen-containing compoundwhich is capable of releasing nitrogen in a form suitable forassimilation by the microorganism. In the organic category, thefollowing compounds can be listed as exemplary nitrogen-containingcompounds which can be used: proteins, acid-hydrolyzed proteins,enzyme-digested proteins, amino acid, yeast 5 3,409,506 6extraefi-aspar'agim, urea, etc. For reasons of econ my. MINERALNu'iinrizre'i 101 i CONCENITRATIVON' origins itis usually preferable toemploy an inorganic compound LITER) such asammonia, ammonium hydroxide,or salts thereof 1 For Good For Poor such'as ammoniurn'phosphate,ammonium citrate, ammo- I? I I t I {Troyvthglpgglal nium sulfate,ammonium acid phosphate", etc.-A'very con- 5 p I v 1 3223 1? iaf venientand satisfactory method of supplying nitrogen is to employ ammoniumhydroxide, ammonium phosphate F830,7 M4 M0 or ammonium acidphosphate,which can be added as the Ken... 1. 0- '"N/ C i sot. o.'25 4 0.05-0.20salt per .se or can be produced in situ 1n the aqueous Mnsm (104 MCfermentation media by bubbling ammonia through'the l0 N tSO 0.5 N/C 85%H3P04 2. 5 N/C broth ,to which phosphoric acid was previously added,thereby forming ammonium acid phosphate. In thisway .gWeightD =(g-l 9.the pH.range of 5.5-7.5 is maintained and the requisite N0 f nitrogen issupplied. Ammonium hydroxide can be supplied to the reactor in amountsof hetweenab ont 0.01 and about 1.0 weight percent, preferablybetweenabout 0.1 and abou't'0.15 weight percent, nitrogen.

addition' to the energy and nitrogen sources, it is also necessary'tosupply requisite amounts of selected mineral nutrients to thefermentation reactor. Thus, potassium, so- 0 dium, iron, magnesium,calcium, manganese, phosphorus and other nutrients are included in theaqueous growth medium. These necessary materials can be supplied'in theand about Q The exaetfltemp'erature, pe forth of their Salts andPreferably their watet'fsothhle upon the specific micnoorganism beingutilized; but, a For example the Potassium can be supplied as P usuallytemperatures of between about 20 C. and about tass'ium chloride,phosphate, sulfate, citrate, acetate, ni- 4 C. are employed, P f bl thef t tion is p In another embodiment of the present invention, it hasbeen found that the use ofminor amounts, e.g., between about 1 and about75 ppm, of azelaic acid in the.aqueous fermentation medium stimulates.ester formation. Preferably, about'SO p.p.m. of azelaic acid isemployed. Az elaic acid (1,7-heptane.dicarboxylic acid) is generallyprepared by the disruptiveoxidation of. ricinol eicacid. Q

The temperature at which. the fermentation reaction bf the presentprocess is carried out can vary between about ttate" Iron and Phosphoruscan be supplied 'th t conducted .at temperatures of between about 25 C.and form of sulfates and phosphates, respectively, e.g., iron about Isuttate a iron Phosphate- Usuauy most of the R The pH of thebiosynthesis bath is generally held at phor-hs Supplied as ammehlum P t.Wheh etther between about 5.5 and about 7.5. -If the pH becomes tooammohlumphosphate or athmomuth held phosphate 15 high, .it can belowered readily by the addition of a s can: Serve a cethblhed Source ofhlttogeh and suitable acid to the fermentation media, e.g., H PO InPhosphgrusv I f i like manner, if the pH becomes toolow, it can beraisedIn accordance with the present process, It has been by the addition of asuitable base, e.g., ammonia orttmfound that by limiting theconcentration of selected numehium hydroxide. v tnent miner-a1 IOIIS 1nthe aqueous nutrient medium to be At the start up f the fermentation,the growth medium row that required for maximum m FE g e t is inoculatedwith a vegetative microbial inoculum. The h metahohe actleh 9 emlereergahlsm can h 'he initial concentration of said inoculum at the.outset of nested to Produce esters from the hydroeerbon feed'fermentation. can vary widely, e.g., from about 0.1 to

lht microorganism cell growth th the e 40 about 1.0 gram per liter oftotal fermentation media. ventional' biosynthesis fermenter, whereinmicroorganisms The fermentation reactor can be Stirred during biosyn;

are aerobically Cultivated, the P y fhhetieh of the thesis by anyconventional means such as a paddle stirrer, eroorgahism is to reproduceitself and thlls y fL as P fi propeller-type stirrer, rockers or otheragitator means llcie-micmofvgahismfiells-eeByepliodllctsthe-ehlosyhthehee-flto-completely admix the oxygen, mineral nutrients;hydroculttyattoht Such as acids and esters are t e kept to 5 carbon andmicroorganism. For example, paddle stirrers a h i However, by alteringthe composlhoh e'f-tthe can be used having a horsepower rating of 1-100,preferaqu'eous mineral salts solution by limiting the concentraably 530, per 1,000 gallons f liquid reactant medium tiondofselected mineralions, .by-products of the biosyn- The liquid residence time incontinuous operations for thesis eultivafion, Such as fatty acid esters?can be t a bacteria such as Micrococcus cerificans (Arthrobacttzr tivelyproduced atthe expense of cellular e 1 5O lll'eafdciens), i.e., thevolume of liquid in the reactor --Morespecifically, it'has beenfound'that 1f the concendivided hythe amount f the material supplied(and tration of the magnesium and/or calcium mineral nutrients productsremoved so as to maintain a constant liquid iS res c e to levels belowthat q r for maxlmum level in the reactor) per hour, is,'in general, 1to 10 hours, cellular growth, esters are formed at the expense of celfbl 1 to 3 hours, more preferably 15 2 hours.

lular production. The magnesium and calcium mineral The present processcan, f course a ,be carried out by nutrient concentrations can berestrlcted Independently or hatch or continuous means.

at the a time- However, t concentration. of the {h3 1 The productsproduced in accordance with the present ne'sium mineral nutrient hasbeen found to be more critical process are primarily acids and estersThe ifi acids than th of ealehhh for the Selective P d o of e P andesters produced will depend.v to a large extent on I11 g n thecohcehtratioh 0f the divalent ealehhh the hydrocarbon feed stockemployed. Thus, for example, (Ca++) ion in the fermentation reactorshould not be if a C16 hydrocarbon is employed as the feed the more thanabout i t Pereent hl ordef to Promote pal acid and ester produced willbe palmitic acid and the p e e en 0f esters and, preferably, W 1 ancetyl palmitate. Mixed hydrocarbon feeds will produce tween about 0.005and about 0.01 weight-percent (0.05- a Spectrum f acids and esters I 0J0grams/liter). The concentration of the divalent mag- The esters producedin accordance with the Present' heshlm E ion in e tetmehtatloh reatftol'should process are essentially present in the aqueous fermentanot bemore than about 002 Weight p e In Order t tion media (extracellular).However,'-some of the esters Pmmoteihe Preduction Of-eSteYS-ahd,epretehahtytwth for-medare held within the microorganism cell itself.tahge between about 0-005 and about Wetght pefcent Esters produced canbe extracted from the fermentation (0.050.20 grams/liter). The followingtable contams'a media. from the concentrated microorganism cells usingtabulation-of the mineral nutrient ions conventionally em- Solvents Suchas acetone aeetone-water.hexane i ployed in-the aqueous growthmediumg-th salts by whichtwee t ei n are conventionally pp and aComparison The esters produced in accordance with the present of theconcentration'of each ion for cellular growthversus I a proc ss areexcellent starting materials for the producs'teFpFod'uetion." 7 tion ofC -C alcohol-s. The esters are readily hydrogenated to alcohols in asimple one-step catalytic process. Moreover, the esters can readily besaponified to produce soap. In addition, the esters can be employed aschemical intermediates or for applications in which the ester is usedper se.

The present process is more particularly described in the followingexamples which are intended as illustrative only since numerousmodifications and variations therein will be apparent to those skilledin the art.

EXAMPLE 1 A 4-liter cylindrical reactor was thoroughly sterilized withsteam. The reactor was equipped with a stirrer having a power rating of40-60 horsepower per 1,000 gallons and with various equipment foroperating the reactor continuously. The sterilized reactor was chargedwith 4 liters of an aqueous salt feed having the composition set forthin Run No. 1 of Table I. Sufiicient normal hexadecane was then chargedto the reactor so that about 1 weight percent, based on salt solutionfeed, was present. Thereafter, Micrococcus cerificans in the form of aninoculum was added to the reactor so that said microorganism was presentin an initial concentration of about 0.5 gram/liter. The temperature ofthe reactor was adjusted to 95 F. and the pH regulated to about 7.0. Airwas introduced into the reactor at a rate of between about 1.0 and about3.0 liters/minute. The aqueous mineral salts solution was added at therate of 2 liters/hour. Normal hexadecane was introduced at a rate of 20grams/hour. A material balance was made at steady state conditions andthe average percent carbon, unaccounted for as carbon dioxide and cells,calculated. This figure indicates the amount of carbon present in theester and acid. In general, greater than 85% of the total acid and esterproduct is present as ester and usually greater than 90% of said totalproduct is ester.

Run 2 was performed in the same manner as Run 1 as was Run 3 with theexception that the concentration of magnesium and calcium salts werechanged. Results are tabulated in Table I.

8 The data contained in Table II demonstrate that the magnesium ionconcentration is effective in regulating the amount of fatty acid esterproduced; but, has no effect on the percent of hydrocarbon converted tocells, carbon dioxide, water and other by-products.

EXAMPLE 3 Growth media containing either 1 weight percent or 2 weightpercent hydrocarbon were prepared having the following composition:

Concentration Component: (grams/ liter) n-Hexadecane 1 10 or 20 K HPO5.0 (NH HPO 10.0 Na SO 0.5

H2O 0.4 FeSO -7 H2O MHSO4'4 H2O NaCl 0.02

Water (suflicient to make a volume of 100 mls.). Con1mercialn-hexadecane containing 1 weight percent Cm u-monoolefin.

After regulating the pH to about 7.8, 100 ml. of each of theabove-prepared media were introduced into separate 500 ml. New Brunswickgyratory incubated shake flasks and the flask contents sterilized byheating at 121 C. for about 15 minutes. Then about 1% (1 ml. per 100 ml.of growth medium) of an 18-hour vegetative inoculum of Micrococcuscerificans (Arlhrobacter ureafaciens) was introduced into each of theflasks. The fermentation media were cultured under shaking conditions(300 r.p.m.) at 30 C. for 48 hours. The pH of the fermentation media atthe end of the 48-hour period was about 5.5.

After 48 hours, the fermentation media were subjected to centrifugationand aliquots of the resultant slurries were extracted with four timestheir volume of acetone. Thereafter, the extracts were concentrated toone-half their volume by heating. The concentrated extracts were TABLEI.-EFFECT OF CATION FEED CONCENTRATIONS ON ACID-ESTER FORMATION AveragePer- Average 85% H3PO K01 NagSOr FeSO4'7 H1O MgSO MnSO4-Hz0 CaClz centCarbon Percent Run Number (g./l.) (g./l.) (g. .ll.) (g./l.) (g./l.)(g./l.) Unaeeounted Hydrocarbon for as CO: Converted and Cal The datacontained in Table I demonstrates that when then cooled to 0 C. so as toprecipitate the esters formed the concentration of the magnesium andcalcium salts were reduced to about 0.2 g./l., the amount of ester andacid produced was significantly increased.

EXAMPLE 2 Runs 4, 5 and 6 were performed in a similar manner to Runs 1through 3 with the exception that the air rate was between about 3.5 andabout 4.0 liters per minute, and the mineral salt concentration for eachof the runs was varied in accordance with the amounts tabulated in TableII.

EXAMPLE 4 A series of runs were performed in an analogous manner asthose performed in Example 3 with the exception TABLE IL-EFFECT OFCATION FEED CONCENTRATIONS ON ACID-ESTER FORMATION Average Per- AverageHJPO] KCl NazSO4 FeSO4-7 H O MgSO MnSO4-H O CaClz cent Carbon PercentRun Number (g./l.) (g.ll.) (g. (g./l.) (g.,l.) (g./l.) (g./l.)Unaceounted Hydrocarbon for as 002 Converted and Cells that about 50p.p.m. of azelaic acid were incorporated into the aqueous growth media.On the average, about 3.2 grams per liter of ester together with about7.6 grams per liter of cells were produced per run.

The data contained in Example 4 demonstrates that the use of minutequantities of azelaic acid in the growth medium increases fatty acidester production by more than 600%.

While there are above described a number of specific embodiments of thepresent invention, it is obviously possible to produce other embodimentsand various equivalent modifications thereof without departing from thespirit of the invention.

Having set forth the general nature and specific embodiments of thepresent invention, the true scope is now particularly pointed out in theappended claims.

What is claimed is:

1. A process for the biosynthetic production of esters, which comprisesaerobically fermenting a hydrocarbon containing from about 1 to 30carbon atoms dispersed in an aqueous growth medium through the action ofa microorganism comprising Micrococcus cerificans, said aqueous growthmedium containing less than the amount required for maximum growth ofsaid Micrococcus cerificans mineral nutrients selected from the groupconsisting of divalent magnesium cation, divalent calcium cation, and acombination thereof, wherein said divalent magnesium cationconcentration is between about 0.005 and about 0.02 wt. percent, andwherein said divalent calcium cation concentration is between about0.005 and about 0.01 wt. percent.

2. A process according to claim 1 wherein said aqueous growth mediumcontains between about 1 and about 75 p.p.m. of azelaic acid.

3. A process according to claim 2 wherein said azelaic acidconcentration is about ppm.

4. A process according to claim 1 wherein said hydrocarbon is a C -Chydrocarbon which has been purified to reduced the level of aromatics tobelow about 0.5 weight percent.

5. A process according to claim 1 wherein said hydrocarbon isn-hexadecane.

6. A process for the biosynthetic production of esters, which comprisesaerobically fermenting n-hexadecane dispersed in an aqueous growthmedium through the action of a microorganism comprisingMicrococcuscerificans, said aqueous growth medium containing less than the amountrequired for maximum growth of;said Micrococcus cerificans, of mineralnutrients selectedfrom the group consisting of divalent magnesiumcation, divalent calcium cation and a combination thereof, said divalentmagnesium cation having a concentration between about 0.005 and about0.02 wt. percent, said divalent calcium cation having a concentrationbetween about 0.005 and 0101 wt. percent, said growth medium alsocontaining between about 1 and about p.p.m. of azelaic acid.

References Cited UNITED STATES PATENTS 2/1965 Davis --3 6/1967 Coty195-3 OTHER REFERENCES LIONEL M. SHAPIRO, Primary Examiner,

